We have constructed putative human artificial chromosomes by combining elements necessary for normal chromosomal function such as human telomeres and origins of DNA replication, with yeast artificial chromosomes containing human Y chromosome centromeres. These constructs have been introduced into mammalian cells by spheroplast fusion and liposome-mediated DNA transfection. Cell clones which are suspected to contain the HAC have been isolated based on G418 resistance and propagated for several passages. We are currently analyzing these clones to determine if the HACs remain intact, extrachromosomal and segregate 1:1 during mitosis. Additional constructs are being developed which include known viral origins of replication and larger centromeric sequences. Constructs have also been developed and are being analysed in which the telomeres are removed and the linear HAC is converted into a circular vector.